DIRECTEUR DE LA PUBLICATION / PUBLICATION DIRECTOR : Bruno David Président du Muséum national d’Histoire naturelle RÉDACTRICE EN CHEF / EDITOR-IN-CHIEF : Laure Desutter-Grandcolas ASSISTANTE DE RÉDACTION / ASSISTANT EDITOR : Anne Mabille ([email protected]) MISE EN PAGE / PAGE LAYOUT : Anne Mabille COMITÉ SCIENTIFIQUE / SCIENTIFIC BOARD : James Carpenter (AMNH, New York, États-Unis) Maria Marta Cigliano (Museo de La Plata, La Plata, Argentine) Henrik Enghoff (NHMD, Copenhague, Danemark) Rafael Marquez (CSIC, Madrid, Espagne) Peter Ng (University of Singapore) Jean-Yves Rasplus (INRA, Montferrier-sur-Lez, France) Jean-François Silvain (IRD, Gif-sur-Yvette, France) Wanda M. Weiner (Polish Academy of Sciences, Cracovie, Pologne) John Wenzel (The Ohio State University, Columbus, États-Unis) COUVERTURE / COVER : Trioxys ulmi Čkrkić & Tomanović n. sp., male fore wing. 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Diffusion – Publications scientifiques Muséum national d’Histoire naturelle CP 41 – 57 rue Cuvier F-75231 Paris cedex 05 (France) Tél. : 33 (0)1 40 79 48 05 / Fax : 33 (0)1 40 79 38 40 [email protected] / https://sciencepress.mnhn.fr © Publications scientifiques du Muséum national d’Histoire naturelle, Paris, 2021 ISSN (imprimé / print) : 1280-9551/ ISSN (électronique / electronic) : 1638-9387 Insights into phylogenetic relationships between Trioxys Haliday, 1833 and Binodoxys Mackauer, 1960 (Hymenoptera, Braconidae, Aphidiinae), with a description of a new species of the genus Trioxys Jelisaveta ČKRKIĆ Andjeljko PETROVIĆ Korana KOCIĆ Željko TOMANOVIĆ Institute of Zoology, Faculty of Biology, University of Belgrade, Studentski trg 16, 11000, Belgrade (Serbia) [email protected] (corresponding author), [email protected], [email protected], [email protected] Submitted on 4 April 2020 | Accepted on 5 August 2020 | Published on 16 March 2021 urn:lsid:zoobank.org:pub:D0C3B192-C95F-4579-857D-E4F9451C6BF3 Čkrkić J., Petrović A., Kocić K. & Tomanović Ž. 2021. — Insights into phylogenetic relationships between Trioxys Haliday, 1833 and Binodoxys Mackauer, 1960 (Hymenoptera, Braconidae, Aphidiinae), with a description of a new species of the genus Trioxys. Zoosystema 43 (8): 145-154. https://doi.org/10.5252/zoosystema2021v43a8. http://zoosystema.com/43/8 ABSTRACT Despite extensive research on the taxonomy and phylogeny of the subfamily Aphidiinae Haliday, 1833, certain questions about the relationships between genera remain unresolved. Genera Trioxys Haliday, 1833 and Binodoxys Mackauer, 1960 are considered closely related, based on morphological and molecular analyses. However, recent studies suggest there is a need for a taxonomic revision of the two genera, since molecular data does not support monophyly of the two groups when a larger KEY WORDS Aphidiinae, number of species is used in the analysis. We examine those relationships using molecular data and Trioxina, including a new species we describe in the present study. Trioxys ulmi Čkrkić & Tomanović, n. sp. Trioxys, is a parasitoid of the Japanese elm aphid (Tinocallis takachihoensis Higuchi, 1972) on elm hybrids Binodoxys, DNA barcoding, (Ulmus x hollandica Mill.). Despite its probable Asian origin, this species has gone undescribed until new species. its accidental introduction to Europe, highlighting the importance of continued research efforts. RÉSUMÉ Aperçu des relations phylogénétiques entre Trioxys Haliday, 1833 et Binodoxys Mackauer, 1960 (Hyme- noptera, Braconidae, Aphidiinae), avec la description d’une nouvelle espèce du genre Trioxys. Malgré des recherches approfondies sur la taxonomie et la phylogénie de la sous-famille des Aphi- diinae Haliday, 1833, certaines questions sur les relations entre les genres demeurent non résolues. Les genres Trioxys Haliday, 1833 et Binodoxys Mackauer, 1960 sont considérés comme étroitement apparentés, d’après des analyses morphologiques et moléculaires. Cependant, des études récentes sug- gèrent qu’une révision taxonomique des deux genres est nécessaire, puisque les données moléculaires ne supportent pas la monophylie des deux groupes lorsqu’un plus grand nombre d’espèces est utilisé MOTS CLÉS Aphidiinae, dans l’analyse. Nous examinons ces relations en utilisant des données moléculaires et en incluant une Trioxina, nouvelle espèce que nous décrivons ici. Trioxys ulmi Čkrkić & Tomanović, n. sp. est un parasitoïde Trioxys, du puceron japonais de l’orme (Tinocallis takachihoensis Higuchi, 1972) sur les hybrides de l’orme Binodoxys, DNA barcoding, (Ulmus x hollandica Mill.). Malgré son origine asiatique probable, cette espèce n’a pas été décrite jusqu’à espèce nouvelle. son introduction accidentelle en Europe, soulignant l’importance des efforts de recherche continus. ZOOSYSTEMA • 2021 • 43 (8) © Publications scientifiques du Muséum national d’Histoire naturelle, Paris. www.zoosystema.com 145 Čkrkić J. et al. INTRODUCTION morphological characters with ImageJ software (Schneider et al. 2012). Morphological terminology follows Sharkey & Trioxys Haliday, 1833 is one of the largest genera within the Wharton (1997). subfamily Aphidiinae Haliday, 1833, with currently over 70 species described worldwide (Yu et al. 2016). Classified in MOLECULAR ANALYSIS the subtribe Trioxina Mackauer, 1961 based on morphology, DNA was extracted from three individual adult parasitoids its main diagnostic characters are accessory prongs on the using the QIAGEN Dneasy® Blood & Tissue Kit (Qiagen Inc., last abdominal sternite, as well as the absence of secondary Valencia, CA, USA) following the manufacturer’s instructions. tubercles on the petiole (Mackauer 1961). The second char- The barcode region of the COI gene was amplified using the acter, secondary tubercles on the petiole, is traditionally used universal primers LCO1490 and HCO2198 (Folmer et al. to separate Trioxys and the closely related genus Binodoxys 1994). DNA amplification was performed in a 20 μl volume, Mackauer, 1960. Recent studies employing morphological containing 1 μl of DNA, 11.8 μl of H2O, 2 μl of High Yield and molecular data show there is no clear distinction between Reaction Buffer A with 1 × Mg, 1.8 μl of MgCl2 (2.25 mM), the two genera, and that there is a need to revise the current 1.2 μl of dNTP (0.6 mM), 1 μl of each primer (0.5 μM) and status of Trioxys and Binodoxys, or at least some of the species 0.2 μl of KAPATa q DNA polymerase (0.05U/μl) (Kapa Bio- within these two genera (Čkrkić et al. 2019). systems Inc., Boston, USA). PCR amplification was performed Japanese elm aphid, Tinocallis takachihoensis Higuchi, 1972 in an Eppendorf Mastercycler® (Hamburg, Germany), using is an aphid of Asian origin, commonly found on Ulmus spp. in the following thermal profile: initial denaturation at 95° C its native area, as well as in Europe, North Africa, and North for 5 min, followed by 35 cycles of 94° C for 60 s, 54° C for America, where it has been introduced (Blackman & Eastop 60 s, 72° C for 90 s and a final extension at 72° C for 10 min. 1994). Other than Ulmus spp., it can be found on Hemiptelea Purification of PCR products and DNA sequencing in both davidii (Hance) Planch. in China and eastern Siberia, and it is direction was performed by Macrogen Inc. (Seoul, Korea). relatively common on bonsai Zelkova serrata (Thunb.) Makino Sequences were edited with FinchTV ver. 1.4.0 (www.geo- in England (Blackman & Eastop 1994). The introduction of spiza.com). CLUSTAL W algorithm integrated in MEGA X T. takachihoensis to Europe has been accidental, and it is prob- software (Kumar et al. 2018) was used to align sequences. Se- able that it came via bonsai plant trading (Petrović-Obradović quences were trimmed to a length of 638 bp. Newly acquired et al. 2018). While this species is not considered a serious sequences in this study are deposited in GenBank. pest, large amount of honeydew deposits can inflict damage Two additional sequences, identical to those of the new on young trees (Petrović-Obradović et al. 2018). species described here, were acquired from the BOLD da- There have been reports of parasitoids from the genus tabase (http://www.boldsystems.org; BIN AAU8586). Ad- Trioxys parasitizing Tinocallis species (Starý 1978, 1987, ditional sequences of Trioxys complanatus Quilis Perez, 1931, 1988; Lumbierres et al. 2005). However, only one parasitoid T. auctus (Haliday, 1833), T. sunnysidensis Fulbright & Pike, (Aphidius sp.) of T. takachihoensis has been recorded so far from 2007, T. parauctus Starý, 1960, T. pallidus (Haliday, 1833), Algeria (Hemidi et al. 2013). Here we report a new species Trioxys sp., Binodoxys angelicae (Haliday, 1833), B. acalephae of